Table 1: Polymerase chain reaction (PCR) conditions for 16S, blaTEM and nim.

PCR conditions 16S (625 bp) blaTEM (618 bp) nim (458 bp)
Initial denaturation 95°C for 5 min 94°C for 5 min 94°C for 10 min
Denaturation 94°C for 1 min 94°C for 1 min 94°C for 30 s
Annealing 55°C for 1 min 62°C for 1 min 62°C for 1 min
Extension 72°C for 1 min 72°C for 1 min 72°C for 1 min
Step 2 to 4 34 cycles 35 cycles 32 cycles
Final extension 72°C for 5 min 72°C for 10 min 72°C for 10 min
DNA template 5 µl 5 µl 5 µl
dNTPs mix, 10 mM 1 µl (200 µM) 2 µl (400 µM) 1 µl (200 µM)
Reaction Buffer 5x 10 µl 10 µl 10 µl
MgCl2 Solution, 25 mM 3 µl (1.5 mM) 4 µl (2 mM) 3 µl (1.5 mM)
Upstream Primer, 10 µM 2.5 µl (0.5 µM) 5 µl (1 µM) 5 µl (1 µM)
Downstream Primer, 10 µM 2.5 µl (0.5 µM) 5 µl (1 µM) 5 µl (1 µM)
Taq polymerase, 5 U/µl 0.25 µl (1.25 U) 0.25 µl (1.25 U) 0.25 µl (1.25 U)
Water for injection 25.75 µl 18.75 µl 20.75 µl
Values in parenthesis are final concentration

16S primers 5'-CAG GAT TAG ATA CCC TGG TAG TCC ACG C-3' and   5'- GAC GGG CGG TGT GTA CAA GGC CCG GGA ACG-3' [12] blaTEM primers 5'-AGA TCA GTT GGG TGC ACG AG -3' and   5'-CAG TGC TGC AAT GAT ACC GC -3' [15] nim primers 5'-ATG TTC AGA GAA ATG CGG CGT AAG CG-3' and   5'-GCT TCC TTG CCT GTC ATG TGC TC-3' [14]

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